CulturingPlatynereisdumerilii

This culture method is based on more
than forty-five years of experience with a continuous culture of Platynereisdumerilii in the
laboratory using a procedure developed and a worm stock founded by Carl
Hauenschild in 1955. Breeding Platynereisdumeriliiis not trivial insofar as a certain minimum equipment is indispensable and as the
culture requires a certain minimum attention and care. Our method requires a
defined organismal community. It contains a mixture
of microorganisms together with Platynereissome of which are
indispensable as food; others may contaminate the cultures and when they
become dominant (e.g. cyanobacteria, fungi or
ciliates) may endanger the whole culture system. Although future students of Platynereis may streamline the culture method and
make it simpler, we will describe the method as it works in our hands.
Additional details can also be found in Hauenschild
& Fischer (1969; see Title page in the right column).

title page of
Hauenschild & Fischer (1969)

2. What you need!

2.1. The wormsIn the following, we will
concentrate on Platynereisdumerilii
. Much of the text can be applied to Platynereismegalops from the East coast of N America, the
first nereidid reared from the egg to sexual
maturity in captivity (E.E. Just, 1922). We have also cultivated P. massiliensis, a species differing largely in
reproductive biology and requiring a different method of procuring offspring
(Schneider et al. 1991). P. bicanaliculatafrom
the northern Pacific Ocean requires colder
water (12 – 14°C; P.C.Schroeder, pers. comm.).

The
safest source for a new culture of P. dumerilii
is an existing laboratory stock. A number of European labs are breeding
strains which mostly originate from Hauenschild’s
original lab strain isolated from the Bay of Naples
in 1953 and 1955. Another safe way is the collection of swarming, “epitokous” (sexually mature) P. dumeriliiin the littoral zone of the coasts of the
Mediterranean or of W Europe (Spain,
France, Great Britain).
What you can catch at the Atlantic coast of N America is not P. dumerilii but P. megalops:
(although the key may tell you so). Collecting “atokous”
(sexually immature) benthic stages of Platynereis
at European coasts may cause problems: There is a sibling species, P. massiliensis, which cannot be distinguished from P.
dumerilii before it has reached sexual maturity
but differs dramatically from the latter species with respect to reproduction
and early development (Hauenschild 1951, Schneider
et al. 1991). So, if you start your culture from an existing stock, you will
probably begin with the benthic worms, whereas you will start with eggs,
embryos and swimming larvae when beginning with your own broods from epitokes caught in the wild. Neither of the two ways will
present serious problems, but your cultures will require food immediately, so
start your cultures of the algae (Tetraselmis
marina) before starting the worm cultures!

P. dumerilii
can be easily transported either as swimming larvae or, more safely, after
metamorphosis into 3-segmented young worms. Worms at the sessile stage should be
allowed to settle for least for a day in the transport container so that they
can build tubes before traveling into which they
can retreat during shipment. The container should be filled to a larger part
with air to provide enough oxygen. Temperature during the transport should
not exceed 20 – 22° C for an extended period. Vacuum bottles are fine
for transporting the swimming early stages but the use of vulcanized fittings
will kill them very effectively!

Natural seawater has a very
complicated composition. We (and H.-D. Pfannenstiel)
have tried to use artificial seawater (Tropic Marin salt mixture) in the Platynereis cultures but found a decrease in
reproductive success. Even though we have not done a systematic study we
would strongly recommend the use of natural seawater. Obtaining natural
seawater of good quality can be surprisingly difficult. You need unpolluted
seawater of full salinity (≥32 per mille salinity); P. d. does
not tolerate estuarine waters. The seawater needs to be filtered to remove
plankton and detritus. Otherwise you cannot store your seawater without
fouling. The best sources of filtered natural seawater are marine stations
which operate aquariums. Seawater can be stored cool (<18°C) and in the
dark (to prevent algal growth) for several months. We keep it in 60 liter polyethylene barrels from which we fill 10 liter glass bottles with an outlet, rubber plug and glass
cock close to the bottom so that particles in our seawater can settle and
don’t make their way into our Erlenmeyer flasks. These glass bottles
should also be kept in the dark. Before use, the natural seawater must be
heated by whatever method to 85°, e.g., in large (3 liter)
Erlenmeyer flasks on a plate, to kill prokaryotes and eukaryotic microorganisms. Avoid higher temperatures, which will
cause some salts to precipitate irreversibly. Note that you should have 24
hours for the "pasteurized" seawater to cool to room temperatures.
If this “pasteurization” is omitted, the community in your
culture boxes will soon run out of control. Food algae can be cultured in
artificial seawater containing certain additives (see below). Postlarval stages and adult worms can be kept in plastic
boxes containing a mixture of 50% natural and 50% artificial (Tropic Marin)
seawater sterilized as described above.Transferring large quantities of seawaterrequires an electricpump.

TABLE I
Composition of 10 liters of artificial seawater:

300
g of Tropic Marin and 10 ml of each of the following 5 stock solutions. Stock solution 4 should be
added after sterilization

Tetraselmis
marina, a sessile green flagellate, can be grown under bright,
daylight-type artificial illumination. The high light intensities required by
T. marina differ from the dim light preferable for the Platynereisculture boxes: If these get too
much light, populations of autotrophic flagellates and cyanobacteria
may explode and require too much oxygen at night. So, don’t place algae
cultures and Platynereis containers too
close together. This should also help contaminations of your alga cultures
with other members of the bottom community of your Platynereis
cultures.

As a culture medium an artificial
seawater with increased nitrate and phosphate content is used (see Recipe in
Table I). This solution is heated to 85° C (and not more) in order to kill
eukaryotic organisms a day before use. Alga culture requires flat-bottom
containers; we use 20 x 20 cm polystyrene freezer boxes containing 500 ml
artificial seawater. We start a new series of algal cultures every week. One
of the boxes of a 14 day-old culture serves as the mother culture for starting
a new series: Most of the medium from this culture is poured into a clean
container for waste water; the bottom layer with the mat of green algae is
rubbed off with a clean brush (no metal parts!). The green suspension is
collected in a small flask from which it must be removed within hours by a
clean Pasteur pipette for seeding the new cultures. All the algae in the two
week old cultures in the other boxes are harvested in the same way and are,
after settling over night, dispensed among the Platynereis
cultures the next day (not later !). Be careful in maintaining an absolutely
clean “germline” separate from the
algae used as food! Clean the empty boxes with hot tap water and distilled
water and once in a while remove calcareous sediment crusts with diluted HCl. To feed the worms weekly, you will need two sets of
algae breeding containers, e.g., 2 series of 8 containers.

A culture of Tetraselmis
marina can be obtained from a laboratory breeding Platynereis,
or from algae stock centers such as the one at SAG Goettingen . Another useful flagellate is
the heterotrophic dinoflagellateOxyrrhisspec. It can be cultivated on
cultures of Dunaliellasalina, an easy-to-grow green flagellate commonly
used in laboratories. Dunaliella and Oxyrrhis are kept in 250 – 500ml Erlenmeyer
flasks. Several drops of an Oxyrrhis culture
can be added to an early culture of Platynereis.
The dinoflagellates feed excessively on tiny fish
food particles (and bacteria living on them?) and on green flagellates which
makes their locomotion sluggish. Juvenile Platynereis
now can grab them and do so especially close to sedimented
fish food particles where Oxyrrhis
accumulate in considerable flocks. The Platynereis
cultures are fed Tetramin® fish food flakes weekly.
The flakes are ground in a mortar and the resulting flour is left swelling in
seawater for an hour. This suspension is then dispensed sparingly among the Platynereis cultures. Beware of overfeeding! Too
much Tetramin will cause the culture to start
fouling. Experiment with how little will suffice to satisfy your worms and
leave the bottom community intact!

Once in a while, the adult worms
may be fed minced spinach leaves. Don’t use chemically polluted spinach
(which means: grow it yourself!). Watch the worms grabbing the leaf fragments
which they also use to adorn their tubes for a while. Spinachis a facultativefoodcomponentforPlatynereis .

Platynereisdumerilii is an inhabitant of the warmer coasts
of Europe. The worms and their embryos can
stand room temperature (one of the outstanding advantages in using this
experimental marine animal!) and we breed them at constant temperature (18 or
19°). Constant temperature can be achieved by professional air conditioners
of the type used in food stores. To avoid fungal contamination you must allow
the damp air to leave the culture room. The cooled air should flow at the
ceiling instead of at the level of any persons sitting and working in the
culture room. An even temperature is necessary to avoid temperature peaks
which might kill the worms; it also promotes development at a repeatable
rate. 15° C is the lower limit for early development to take place in our
material. Be sure that the capacity of your air conditioner is sufficient to
overcome the summer peak temperatures outdoors: Hot days are the ggreatest threat to your cultures!
To breed Platynereis
successfully, you need to control the light regime. Each worm spawns only
once in its life: both reaching maturity and the spawning event are
synchronized by a lunar light cycle. You thus need artificial illumination
exclusively in your culture room with a timer switching all the lights on and
off in a daily regime. Maintain your worms under "summer
conditions", i.e., 16h of light and 8h of absolute darkness. In
addition, your worms need a “moon”, a low-light lamp (we use a 15
W bulb) which is switched on for a period of seven days and nights to give one
week of “full moon-sensation” in a 28-day cycle. Errors in the
day-night- and lunar light cycles of your culture room have long-term
negative effects upon your breeding success, so keep untrained people away
from playing with your light switches and window shades! Make a chart of the
moon phases on a calender sheet and mark the start
of collection of the male and female epitokes
(starting six days after the moon phase).

Calender with indications of the end
of the moon phase ("Mondaus"),
the start of breeding ("Zucht") and the
change of water (WW) in the polystyrene boxes (every 14 days).

2.5 Culture setup

The adult Platynereis
worms are kept in 20 x 20 cm polystyrene boxes in 500 ml of
“pasteurized” seawater. Here, they build their tubes,
preferentially in the corners of the box and/or parallel and apposed to neighbor tubes. Only transparent boxes will allow you to
illuminate and watch the worms in both incident and transparent light.
Aeration of the seawater is achieved by glass tubes entering through a hole
in the center of the box lid; clean these glass
tubes once in a while with a pipe cleaner. Use a high-volume, low-pressure
aquarium pump. What you need is a gentle stream of air which by permanent
bubbling essentially promotes convection of the seawater. A strong stream of
air will cause too much evaporation which will raise the salinity too much
over the two weeks between water changes. For the same reason, the lid of the
box should fit tightly. The upper parts of the culture box
walls sometimes need cleaning in order to avoid the formation of films of
algae and cyanobacteria . Otherwise some of your worms will climb up the walls
and try to settle there where they will eventually dry out. Rinse away such
climbers before wiping off the alga film which is done with a clean towel (washed without soap and detergent!).Seawater is exchanged by simply
pouring out the used water into a glass bowl and replacing it with 500ml of
freshly “pasteurized” seawater, perhaps after cleaning the walls
of the boxes. Some worms may have tried to escape: bring them back with your
pipette.
The worms are fed once a week, after exchanging their seawater.

Culture
setup of the Platynereis stock at the GießenUniversity. Photograph by Christina
Schmidt.

2.6 Handling the worms

Platynereisdumerilii should not be handled with forceps.
We handle them with stainless steel needles and self-made pipettes. The tips
of commercial Pasteur pipettes are too narrow and their glass is too
thin-walled. Take a 25 cm piece of lab glass tubing (about 7 mm outer diam.) and fire-polish both ends. Heat the middle of the
glass tube in the flame of a Bunsen burner and pull a symmetric constriction
with an inner width of about 3 mm. Cut the constriction in the middle,
fire-polish the cuts and fit a rubber cap on your new pipette (don´t use vulcanized (colored)
rubber, poison!). Silicone caps may be too stiff. With a needle or with your
fire-polished pipette you can easily chase a worm out of its tube by gentle
poking. You can easily pull up the worm into the pipette by sudden suction.
But be sure to expel quickly or they will settle and stay there. Remove Platynereis tube silk and adhering faeces from
your pipettes with a pipe cleaner once in a while and rinse them with hot tap
water.

2.7 Observing Platynereis

For studying Platynereis,
you will need a dissecting stereomicroscope with a long distance objective
lens, an indirect or transillumination stage and a
magnification range between 6.3 x and 40x. A
long-distance objective lens is required because you will need to observe the
worms in their tubes. So, you must put the whole culture box under the
dissecting scope and you need a working distance of 6 cm or more. A transillumination stage is necessary because you will
need sufficient image contrast when examining early development stages at
high magnification. For working with incident light, a fiber
light source is preferable. For watching cleavage and larvae you will need a
compound microscope, preferably with Differential Interference Contrast
(“Nomarski optics”; see Development).

2.8 Breeding and raising PlatynereisdumeriliiReproducing under laboratory conditions
is the most important feature of Platynereisdumerilii. Worms can be continuously available at
every stage of development and of sexual maturation. However, breeding this
animal in the laboratory requires continual attention.

Thanks to the artificial moonlight
cycle, most of your worms will become mature during a ten-day period with a
maximum at about 16 days after switching off the “moonlight”. If
you would leave the sexually mature worms in their boxes, they might lack a
mature partner (sexual maturity only lasts one day/night!). Or, if it mates
successfully, the eggs will be scattered all over the box and have to be
fished out, a tedious piece of work. But the worst result would be heavy protist contamination of the jelly surrounding the fertilized
eggs. Thus, the maturing worms need to be
segregated every second day during the monthly maturation period, before they
swarm and spawn. Isolating and rinsing them is the best chance to start clean
cultures again! You can recognize maturing Platynereis
in their tubes because of their empty gut, the decreasing number of white
pigment cells in the trunk and the increasing
opaqueness of the worms as the gametes fill the body cavity. As a beginner,
you may have problems to discriminate between females and males: As sexual
maturation proceeds, the females will become lemon-colored,
with lemon-colored eggs visibly filling the body
cavity. The males will have a creamy white anterior, and the larger posterior
portion of the trunk will be red from the increased number of blood vessels.
Poke them out of their tubes and separate the sexes into two large glass
bowls with 200 – 400 ml of seawater and aeration where they will
rebuild tubes and mostly not spawn out before you place the sexes together. Change
the seawater in these bowls every day. Once fully mature, the worms will
leave their tubes and will search for a partner by rapid swimming during
the night. When you check the epitokes in the
morning, you will instantaneously recognize those which have tried to spawn
the preceding night: They have left their tubes or, after their nocturnal
swarming, have procisionally crept back into a
tube, often with their tails waving slowly outside the tube opening.

The following morning, bring one or
a few mature male(s) and female(s) together in one glass finger bowl
(crystallizing bowl) with 20 - 30 ml of seawater. The worms will recognize
each other’s sex pheromones immediately and discharge their gametes in
a minute in a rapid dance (film C1577, Institut für den Wissenschaftlichen Film, Göttingen;
<www.iwf.de>). The eggs will immediately be fertilized and sink to the
bottom. Change the supernatant about two minutes after sperm ejection. In the
course of a few minutes, the fertilized eggs will discharge their cortical
jelly precursor material and become sticky. Swelling of the egg jelly is an
absolutely certain indicator of successful insemination. Now distribute the
brood with a fresh Pasteur pipette into 3 - 4 glass bowls in order to avoid
crowding and oxygen depletion and let them develop without aeration. The
mated females are discarded. The males can be used for additional matings at the same occasion. Seawater from a bowl with a
freshly mated pair will readily induce spawning also in an isolated female
which indicates the chemical nature of the spawning signals exchanged between
the partners. If you are short of mature worms, you can store ripe males in
the refrigerator and use them, mostly with success, the next day. Artificial
assistance in spawning is possible in a male dancing but unwilling to spawn:
just press it a little with a sufficiently clean finger tip (not possible for
smokers): a cloud of sperm will come free from the tail. There is little help,
however, for females unwilling to spawn: if you try to remove the eggs from
the coelom you will find them sticky and not ready
for fertilization.

If you don’t separate the
sexes in your collection of matures before the spawning night, you may get
lots of fertilized eggs as well. However, you won’t know the exact time
of fertilization and you cannot keep them as clean as when they spawn by
pairs as described above.

Now place all the bowls containing
eggs in transparent containers and let them develop till the other morning;
then place these boxes on a table and illuminate them from one side (e.g.,
with a desk lamp). The trochophore larvae hatched
from the egg jelly will all assemble towards the upper water rim at the
illuminated side by a positive phototactic
reaction. Here you can collect them with a fresh Pasteur pipette and transfer
them into a fresh bowl thereby isolating them
from decaying egg jelly, from damaged embryos and larvae and from any protists which may have made their way into the bowl.
Don’t try to collect the last larvae from the bottom: you may
induce decay in your collection of healthy larvae.

The trochophores may be left for
another 2 – 3 days in their glass bowl. On their 7th day of life at the
latest you have to transfer them into their penultimate environment: Glass petri dishes 15 – 20 cm in diameter, filled with
about 8mm of seawater. Place these dishes at a dim place in your culture
room. The brood has reached the stage of a three-segmented young worm still
capable of swimming by ciliary motion (“nectochaeta larva”). Now, these tiny worms start to
build their first tubes and, after about 7 days, start feeding. Spread a few
drops of your harvested Tetraselmis algae
over the dish bottom, add a few drops of the lightly milky supernatant of
your Tetramin® powder suspension and, if possible,
a few drops of your Oxyrrhis culture.
Successful feeding such a young culture is the ultimate test of your skill:
all too often these young cultures are overfed and suffocate. So, check their
condition dayly using your dissecting microscope,
and if only a few algae etc. are left with many hungry young worms, then you
can add a little food again. These petri dishes
should not look green but just a slightly greenish tinge!

After four to six weeks of living in Petri dishes, the young
worms are gently brushed off using a metal-free brush or pencil: rice
straw/bamboo pencils sold at shops with artists’ equipment are perfect
for this purpose. The young worms are now placed in their definitive home,
the polystyrene box. Survival rates of the individual broods are very
different, so you may start a new culture box with one or several broods.
Leave such a new culture un-aerated for a couple of days until the young
worms will have firmly settled and don’t change the seawater for about
six weeks. Label the cultures with the date of spawning and any other
information you need..

EpitokousPlatynereis-male
with an opaque (sperm-filled) anterior and muscular posterior part with
paddle-shaped parapodia. Note the rosette at the
rear end serving as a sperm-sprinkler. Photograph by C. Schaub.

TABLE II - Schedule of duties in the culture room

Monday:
-check the thermograph (replace recording chart)
-check the presence of epitokes in the culture
boxes*
-select swarming epitokes from the bowl for
breeding*-change
the water in the bowls of epitokes*
-pasteurize seawater for coming thursday
-feed spinach (small amount!)

Wednesday:
-check the presence of epitokes in the culture
boxes*-select
swarming epitokes from the bowl for breeding*-change
seawater in bowl of epitokes *-collect
swimming trochophores (yesterday´s
breeding)
-harvest Tetraselmisalgae from the
boxes (leave it
standing till tomorrow)
-start a new culture of Tetraselmis-transfer a small volume of Dunalliela
stock to a small
Erlenmeyer flask with artificial seawater
-transfer a small volume of Oxyrrhis to last
week´s
culture of Dunalliela-clean aeration glass tubes with pipe cleaner
-clean polystyrene boxes and Erlenmeyer flasks with
diluted HClThursday:
-the seawater should be exchanged every 14 days
(consult the calender)
-feed young stages with supermatant of Tetraselmis
-feed the worms with sedimentedTetraselmis-grind
Tetramin® fish food in a mortar and add some
seawater-moderately
feed the worms with the soaked fish food-collect
swimming trochophores (yesterday´s
breeding)
-nectochaetes can be transferred from finger bowls
to small petri dishes (add 10 drops of Oxyrrhis)
-check the aeration system in the boxes after
exchanging the seawater and feeding

Friday:
-check the presence of epitokes in the culture
boxes*-select
swarming epitokes from the bowl for breeding*-change
seawater in bowl of epitokes *-at
the age of 4-6 weeks the juveniles should be
transferred from the petri dishes to
polystyrene boxes,
but leave them unaerated for several days.-once
the animals in a box get fewer (due to the constant losses of epitokes) they may me put together
with individuals of approximately the same age.

* starting one week after the moon phase
** to start or terminate the 7-day moon phase

3. What you don’t need

a) No chemicals, no soap, no detergent, no felt pens in the
culture room!
Never use soap or other detergent when you wash the culture boxes, the
glassware, the boxes for algae culture or the towels used for cleaning the
walls of the boxes. Hot water or, if necessary, diluted HCl
is our method of cleaning. Rinse with distilled water and let these items dry
completely before use! Paper towels are sometimes insecticide-treated, so
don’t use them. Students tend to label your glassware with felt pens:
this is poison as soap and detergents. Check where the pump takes the air
from which is blown into your cultures. Make sure that it will not spill oil
into your cultures.

b) No exchange of glass and plastic ware with other labs: Work
with your own set and never mix it with glassware from other labs.

c) No salt crusts! Seawater aggressively corrodes all metals
and, upon evaporation, leaves crusts of wet salt. So, be tidy
and immediately clean the place where you have spilled seawater.
Especially endangered are optical equipment and
books or other paper (e.g., research notes). No rusty equipment whatsoever in
your culture room!

d) No irregular schedule!Maintain a
regular weekly schedule of changing seawater, feeding, start and harvesting
of algae cultures, “moon rise” and “moon set”,
sterilization of seawter (which requires a day for
cooling down to your culture room temperature !) etc. A regular schedule
helps you remember to perform the necessary procedures. If you have done your
duty for a while, your cultures can, on the other hand, stand your absence
for two weeks or so, as long as temperature control, day- and moonlight
cycles and your air pump will continue to work reliably.